Diaphanous family of formin protein is important for nucleation and linear elongation and bundling of actin filaments, and operate downstream of p21 small GTPases of the Rho family. The roles of formins and related proteins during motility and invasion process of breast cancers are not clearly known, especially in relation to the downstream specificity of the RhoGTPase isoforms RhoA versus RhoC. Here, we will develop new fluorescent biosensors for Diaphanous family of formins pertinent to this important motility regulatory pathway, useful for molecular and cellular analysis of cancer cells during invasion. The current limitations to better understanding the molecular regulatory pathways controlled by formins including the Diaphanous related formins (DRF) that control and coordinate the motile mechanisms including actin cytoskeleton reorganization, protrusions, and invasive matrix degradation, are due to lack of sophisticated imaging technologies capable of specifically targeting these important signaling nodes in living cells. We will use these new biosensors in combination with our proven biosensors for the Rho GTPases to directly probe the mechanisms these mammalian Diaphanous formin mDia1 and mDia2 isoforms regulate during cancer invasion in invadopodia protrusions of metastatic mammary adenocarcinomas.